Refining the phenotype of alpha-1a Tubulin (TUBA1A) mutation in patients with classical lissencephaly.

Mutations in the alpha-1a Tubulin (TUBA1A) gene have recently been found to cause cortical malformations resemblant of classical lissencephaly but with a specific combination of features. To date, TUBA1A mutations have been described in five patients and three foetuses. Our aims were to establish how common TUBA1A mutations are in patients with lissencephaly and to contribute to defining the phenotype associated with TUBA1A mutation. We performed mutation analysis in the TUBA1A gene in 46 patients with classical lissencephaly. In 44 of the patients, mutations in the LIS1 and/or DCX genes had previously been excluded; in 2 patients, mutation analysis was only performed in TUBA1A based on magnetic resonance imaging (MRI) findings. We identified three new mutations and one recurrent mutation in five patients with variable patterns of lissencephaly on brain MRI. Four of the five patients had congenital microcephaly, and all had dysgenesis of the corpus callosum and cerebellar hypoplasia, and variable cortical malformations, including subtle subcortical band heterotopia and absence or hypoplasia of the anterior limb of the internal capsule. We estimate the frequency of mutation in TUBA1A gene in patients with classical lissencephaly to be approximately 4%, and although not as common as mutations in the LIS1 or DCX genes, mutation analysis in TUBA1A should be included in the molecular genetic diagnosis of classical lissencephaly, particularly in patients with the combination of features highlighted in this paper.

Location and type of mutation in the LIS1 gene do not predict phenotypic severity.

BACKGROUND: Lissencephaly is a neuronal migration disorder leading to absent or reduced gyration and a broadened but poorly organized cortex. The most common form of lissencephaly is isolated, referred as classic or type 1 lissencephaly. Type 1 lissencephaly is mostly associated with a heterozygous deletion of the entire LIS1 gene, whereas intragenic heterozygous LIS1 mutations or hemizygous DCX mutations in males are less common. METHODS: Eighteen unrelated patients with type 1 lissencephaly were clinically and genetically assessed. In addition, patients with subcortical band heterotopia (n = 1) or lissencephaly with cerebellar hypoplasia (n = 2) were included. RESULTS: Fourteen new and seven previously described LIS1 mutations were identified. We observed nine truncating mutations (nonsense, n = 2; frameshift, n = 7), six splice site mutations, five missense mutations, and one in-frame deletion. Somatic mosaicism was assumed in three patients with partial subcortical band heterotopia in the occipital-parietal lobes or mild pachygyria. We report three mutations in exon 11, including a frameshift which extends the LIS1 protein, leading to type 1 lissencephaly and illustrating the functional importance of the WD domains at the C terminus. Furthermore, we present two patients with novel LIS1 mutations in exon 10 associated with lissencephaly with cerebellar hypoplasia type a. CONCLUSION: In contrast to previous reports, our data suggest that neither type nor position of intragenic mutations in the LIS1 gene allows an unambiguous prediction of the phenotypic severity. Furthermore, patients presenting with mild cerebral malformations such as subcortical band heterotopia or cerebellar hypoplasia should be considered for genetic analysis of the LIS1 gene.

In vivo and in vitro effect of glucocorticoids on fibroblast growth factor (FGF)-2 and FGF receptor 1 expression.

In order to clarify the physiological function of fibroblast growth factor (FGF-2) in the adrenal medulla the regulation of FGF-2 and FGF receptor 1 (FGFR1) was studied in vitro and in vivo in response to glucocorticoids. To assess the effects of glucocorticoids, in vivo extracts of adrenal medulla and adrenal cortex were analyzed by RNase protection assay and Western blot analysis. PC12 cells were chosen as a model system to study the effects of glucocorticoids in vitro. In PC12 cells, dexamethasone (DEX) was found to stimulate dramatically the expression of both FGF-2 mRNA and protein. Western blot analysis revealed that exclusively the 21-kDa FGF-2 isoform was enhanced. In contrast to the FGF-2 mRNA level FGFR1 was not affected by treatment with glucocorticoids. In vivo FGF-2 mRNA level and 21-kDa FGF-2 isoform level are significantly enhanced in the adrenal medulla 24 h after DEX injection. In vivo application of DEX leads to an increase of the medullary and cortical FGFR1 transcript levels. Glucocorticoid effects on FGF-2 expression were not found in adrenal cortex, heart, skeletal muscle, and kidney, respectively, in vivo and in L6 rat myoblasts in vitro. In addition to adrenal medullary cells glucocorticoids elevated the FGF-2 mRNA and protein level also in vivo in the brain and in vitro in immortalized Schwann cells. The present results suggest that the 21-kDa FGF-2 isoform mediates a physiological function specific for neuronal tissue which is modulated by glucocorticoids.

Duck hepatitis B virus: DNA polymerase and reverse transcriptase activities of replicative complexes isolated from liver and their inhibition in vitro.

The duck hepatitis B virus (DHBV)-associated activities of reverse transcriptase and DNA polymerase and their inhibition in vitro were studied. Replicative complexes (RCs) were isolated from DHBV-infected liver by gel chromatography followed by sucrose gradient centrifugation. The RCs were detected by dot blot hybridization, using radiolabeled cloned DHBV DNA as a probe, and by the incorporation of 32P-TTP in the presence of dATP, dCTP, dGTP, and Mg2+ (endogenous DNA polymerase activity). The endogenous DNA polymerase activity associated with RCs was further studied using exogenous templates: reverse transcriptase and DNA polymerase activities were demonstrated using as substrates 32P-TTP and poly(rA) p(dT)12 or poly(dA) p(dT)12-18, respectively. Both activities were biochemically characterized. Their inhibition by various antiviral agents was studied in vitro: actinomycin D, ara-ATP, aphidicolin, suramin, chloroquin, and phosphonoformate. Among these, suramin, chloroquin, phosphonoformate, and ara-ATP were shown to be potent inhibitors of viral reverse transcriptase and DNA polymerase. Studies are now in progress to establish their antiviral activity in vivo.

Spot-blot hybridization assay for the detection of hepatitis B virus DNA in serum: factors determining its sensitivity and specificity.

Factors determining the sensitivity and specificity of the spot-blot hybridization technique for the detection of hepatitis B virus DNA in serum were systematically investigated. Methods for pretreatment of serum samples, mode of application of the samples to the transfer membranes, blot treatment and hybridization conditions were all found to affect the sensitivity of the assay. The optimum hybridization procedure was found to be incubation of serum samples with salt, NaOH, formaldehyde and detergent, followed by spot application of the samples. This method specifically detected hepatitis B virus DNA in serum with a sensitivity 5 to 15 times higher than the presently used assay procedures.

Hepatocellular carcinoma and hepatitis B virus infection: molecular evidence for monoclonal origin and expansion of malignantly transformed hepatocytes.

The clonality of tumor cells was studied in a patient with metastasizing hepatocellular carcinoma (HCC). Using hepatitis B virus (HBV) DNA as a genetic marker, the pattern of integration of viral DNA into the tumor cell genome was determined by Southern blot analyses of DNAs extracted from different HCC lesions in the liver and both lungs. All tumor tissues examined were found to have viral DNA integrated into the same site(s) of the cellular genome. This finding provides direct molecular evidence for a monoclonal origin and expansion of malignantly transformed hepatocytes during tumor growth and metastasis. This characteristic is similar to other human cancers associated with viral infections, such as adult T-cell leukemia, Burkitt's lymphoma, or cervical cancer, and is important for our understanding of viral oncogenesis in man.